Volume 149, Issue 4, Pages 795-806 (May 2012) RTEL1 Dismantles T Loops and Counteracts Telomeric G4-DNA to Maintain Telomere Integrity  Jean-Baptiste Vannier, Visnja Pavicic-Kaltenbrunner, Mark I.R. Petalcorin, Hao Ding, Simon J. Boulton  Cell  Volume 149, Issue 4, Pages 795-806 (May 2012) DOI: 10.1016/j.cell.2012.03.030 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Telomere Defects Resulting from Conditional Deletion of Mouse RTEL1 (A) RTEL1 genotyping PCR on DNA derived from MEFs of the indicated genotypes. PCR products: WT, 571 bp; flox, 812 bp; null allele, 777 bp. (B) Immunoblots to monitor loss of RTEL1 upon Cre-treatment of RTEL1F/+ and RTEL1F/− MEFs. Tubulin WB as loading control. (C and D) Telomere FISH to monitor telomere integrity upon loss of RTEL1, 96 hr after Cre-treatment. Representative low (C) and high (D) magnification images of chromosomes from WT and RTEL1−/− MEFs. Three classes of telomere dysfunction are observed in RTEL1−/− MEFs: telomere heterogeneity, red; telomere loss, yellow; telomere fragility, pink. (E) Quantitative telomere FISH analysis to monitor telomere length in RTEL1+/− and RTEL1−/− MEFs (significance: one-way ANOVA, p < 0.001). (F and G) Quantification of (F) telomere loss and (G) telomere fragility per metaphase before and 96 hr post Cre-treatment in MEFs of the indicated genotype.Error bars indicate ± standard error of the mean (SEM) from at least 50 metaphases. See also Figure S1. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 TCs Rapidly Accumulate upon Conditional Deletion of Mouse RTEL1 (A) Schematic of the method employed to detect TCs by Phi29 rolling circle amplification with a telomere-specific primer. (B) Phi29-dependent TCs in NIH 3T3 control, TRF2ΔB-overexpressing cells, and VA13 ALT cells. (C) Linear TRFs and Phi29-dependent TCs detected 96 hr post Cre-treatment of RTEL1F/− MEFs. VA13 ALT cells were used as control for TC amplification. (D) Graphical representation of levels of linear TRFs and Phi29-dependent TCs detected 96 hr post Cre-treatment of RTEL1F/− MEFs untreated or treated with ExoV. (E) Graphical representation of the fold induction of Phi29-dependent TCs in RTEL1F/+ and RTEL1F/− MEFs at the indicated time points post-treatment with Cre. VA13 ALT cells were used as control for TC amplification. Error bars indicate ± SEM from at three independent experiments. See also Figure S2. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Blocking DNA Replication on the Telomere Phenotype of RTEL1-Deficient Cells (A) Representative images of telomere fragility in RTEL1−/− MEFs as assessed by telomere FISH. Quantification of fragile telomeres per metaphase in RTEL1F/+ and RTEL1F/− MEFs in the presence or absence of the replication inhibitor aphidicolin (Apd; 0.2 μM) before and 96 hr after Cre-treatment are shown. Error bars indicate ± SEM from at least 50 metaphases. (B) Phi29-dependent TCs in RTEL1−/− MEFs in the presence or absence of the replication inhibitor aphidicolin (Apd; 5 μM). (C) Quantification of the fold induction of Phi29-dependent TCs in RTEL1−/− MEFs subject to the indicated treatments (no treatment, −; aphidicolin, Apd; Hydroxyurea, HU). (D) Quantification of telomere loss per metaphase in RTEL1F/− MEFs in the presence or absence of aphidicolin (Apd; 0.2 μM) before and 96 hr after Cre-treatment (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Effect of SLX4 Downregulation on the Telomere Phenotype of RTEL1-Deficient Cells (A) Phi29-dependent TCs in RTEL1−/− MEFs subjected to control or SLX4 shRNAs. (B) Quantification of the fold induction of Phi29-dependent TCs in RTEL1−/− MEFs subjected to control or SLX4 shRNAs. (C) Quantification of telomere loss per metaphase in RTEL1F/− MEFs subjected to control or SLX4 shRNAs (two independent SLX4 shRNAs: n°1 and n°2) before and 96 hr after Cre-treatment (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from three independent experiments. (D) Quantification of fragile telomeres per metaphase in RTEL1F/− MEFs subjected to control or SLX4 shRNAs before and 96 hr after Cre-treatment (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. See also Figures S3 and S4. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Effect of MUS81, ERCC1, and SLX1 Downregulation on the Telomere Phenotype of RTEL1-Deficient Cells (A) Phi29-dependent TCs in RTEL1−/− MUS81−/− double-knockout MEFs and controls. (B) Phi29-dependent TCs in RTEL1−/− MEFs subjected to control, SLX4, SLX1, or ERCC1 shRNAs (two independent shRNAs for each: n°1 and n°2). (C) Quantification of the fold induction of Phi29-dependent TCs in RTEL1−/− MEFs subjected to control, SLX4, SLX1, or ERCC1 shRNAs. (D) Quantification of telomere loss per metaphase in RTEL1−/− MEFs subjected to control, SLX1, or ERCC1 shRNAs (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. (E) Quantification of fragile telomeres per metaphase in RTEL1−/− MEFs subjected to control, SLX1, or ERCC1 shRNAs (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. See also Figure S5. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Effects of BLM and the G-Quadruplex-Stabilizing Agent TMPyP4 on RTEL1−/− MEFs for TC Formation, Telomere Loss, and Telomere Fragility (A) Phi29-dependent TCs in TRF1−/−, BLM−/−, and RTEL1−/− MEFs. (B) Quantification of telomere loss per metaphase in BLM+/+RTEL1F/− and BLM−/−RTEL1F/− MEFs before and 96 hr post-treatment with Cre (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. (C) Quantification of fragile telomeres per metaphase in BLM+/+RTEL1F/− and BLM−/−RTEL1F/− MEFs before and 96 hr post-treatment with Cre (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. (D) Quantification of the fold induction of Phi29-dependent TCs in RTEL1−/− MEFs treated for 72 hr after Cre with TMPyP4 (10 μM), compared to untreated cells and cells treated with aphidicolin (Apd, 0.5 μM). Error bars indicate ± SEM from at least 50 metaphases. (E) Quantification of telomere loss per metaphase in RTEL1F/− and RTEL1−/− MEFs treated for 72 hr after Cre with TMPyP4 (10 μM), compared to untreated cells (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. (F) Quantification of fragile telomeres per metaphase in RTEL1F/− and RTEL1−/− MEFs treated for 72 hr after Cre with TMPyP4 (10 μM), compared to untreated cells (significance: one-way ANOVA, p < 0.05). Error bars indicate ± SEM from at least 50 metaphases. See also Figure S6. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Model of the Dual Role of RTEL1 in Disassembling T Loops and Suppressing Telomere Fragility (A) RTEL1 disassembles T loop secondary structures to allow replication of the chromosome end. In the absence of RTEL1, persistent T loops are inappropriately resolved by the SLX4 nuclease complex (SLX1- and XPF-dependent manner), resulting in the formation of TCs and telomere loss. (B) RTEL1 and BLM function in distinct pathways to suppress G4-DNA structures to facilitate replication of the telomere. In the absence of RTEL1, G4-DNA structures are a major source of telomere fragility. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S1 Telomere Phenotypes upon Conditional Deletion of RTEL1, Related to Figure 1 (A) IF-FISH to monitor DNA damage induced in RTEL1F/+ and RTEL1F/− MEFs 96 hr following Cre-treatment. Quantification of the percentage of metaphases with > 2 telomere dysfunction-induced foci (TIFs). Cells of the indicated genotype were stained for 53BP1, telomeric repeats (PNA probe) and counterstained with DAPI. Error bars indicate ± SEM from three independent experiments. (B) Quantification of the percentage of metaphases with > 1 telomere fusion in RTEL1F/+, RTEL1F/−, and TRF1F/F MEFs 96 hr following Cre-treatment. Error bars indicate ± SEM. from three independent experiments (C) Phase-contrast images of primary RTEL1+/− and RTEL1−/− MEFs stained for SA-β-galactosidase at day 8 after Cre. Senescent cells contain blue stain. (D) Quantification of chromosome breaks in RTEL1F/+ and RTEL1F/− MEFs 96 hr following Cre-treatment. Representative images of broken chromosomes in RTEL1−/− MEFs. Error bars indicate ± SEM from at least 50 metaphases. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S2 RTEL1 Antagonizes T Loop Structures in an ATPase-Dependent Manner In Vitro, Related to Figure 2 (A) Schematic showing the steps to generate a T loop structure in vitro. (B) Immunoblot to examine the Tet-induced expression of WT and K48R mutant RTEL1 in HEK293 cells. (C) T loop assays were performed with labeled telomere fragments with 3′ or 5′ single-stranded overhangs in extracts from cells induced to express WT or K48R mutant RTEL1. Strand invasion of the labeled telomere fragment into the plasmid with telomere repeats generates a T loop containing plasmid that migrates slowly on the gel compared to the labeled telomere fragment. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S3 Cell-Cycle Profiles and Telomere-Length Heterogeneity of RTEL1−/− MEFs Treated with Control and SLX4 shRNA, Related to Figure 4 (A) FACs analysis of cells of the indicated genotype. Shown is the percentage of cells in G1, S, and G2 phases of the cell cycle. (B) Quantitative telomere FISH analysis to monitor telomere length in RTEL1−/− MEFs stably expressing Control (Ctl) and SLX4 shRNA (n°1 and 2) before and 96 hr following treatment with Cre. Telomere intensity is a measure of telomere length. Error bars indicate ± SEM from at least 50 metaphases. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S4 Immunoblots of the Different Knockout Cells, Related to Figure 4 (A) WB from the protein extracts of RTEL1F/− MEFs stably expressing two different shRNAs (1 and 2) against SLX4. (B) WB with anti-XPF antibody from the protein extracts of RTEL1F/− MEFs stably expressing two different shRNAs against ERCC1/XPF (1 and 2) (Niedernhofer et al., 2006). (C) IP-WB from protein extracts of RTEL1F/− MEFs stably expressing two different shRNAs against SLX1 (1 and 2). 1% of the input was used to run a WB with anti-tubulin antibody as loading controls. (D) Immunoblots for TRF1 in TRF1F/F MEFs not infected (−) and 96 hr after Cre-infection (+). TRF1 runs at 56 kDa. A cross-reacting band indicates equal loading (∗). (E) Immunoblots for BLM in RTEL1F/F BLM+/+ and RTEL1F/F BLM−/− MEFs. BLM runs at 200 kDa. A cross-reacting band indicates equal loading (∗). (F) Immunoblots for MUS81 in RTEL1F/F MUS81+/+ and RTEL1F/F MUS81−/− MEFs. MUS81 runs at 70 kDa. Multiple cross-reacting bands indicate equal loading (∗). Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S5 Replication Inhibition and SLX4 Downregulation in ALT and TRF2ΔB-Overexpressing Cells on TC Amplification, Related to Figure 5 (A) Phi29-dependent TCs in VA13 ALT cells treated with aphidicolin (Apd, 0.5 μM), control or 4 independent shRNAs (SLX4 sh1-4). Quantification of the levels of TCs following the indicated treatments relative to untreated VA13 ALT cells (−). (B) Phi29-dependent TCs in NIH 3T3 cells overexpressing TRF2 or TRF2ΔB treated with aphidicolin (Apd, 0.5 μM), control or SLX4 shRNA. Quantification of the levels of TCs following the indicated treatments relative to untreated TRF2ΔB cells. Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S6 Telomere-Sister Chromatid Exchanges in RTEL1-Proficient and -Deficient MEFs, Related to Figure 6 T-SCE frequency was measured by CO-FISH to estimate the frequency of telomere recombination in RTEL1f/+ and RTEL1f/− MEFs before and 96 hr following treatment with Cre. Shown is the fold induction of T-SCE in the indicated genotypes relative to RTEL1f/+ before Cre. Representative images of no T-SCE (upper image) and T-SCEs (low image) are shown. T-SCE events are marked in the lower panel by (∗). Cell 2012 149, 795-806DOI: (10.1016/j.cell.2012.03.030) Copyright © 2012 Elsevier Inc. Terms and Conditions